Ribavirin-induced hemolytic anemia is the main cause of discontinuation of the combination therapy with alpha-interferon-2b and ribavirin for the treatment of hepatitis C virus (HCV) infection. The determination of intracellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, which could be caused by the accumulation of these molecules within the erythrocytes. In this work, we simplified and validated a previously developed assay method, to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a five-fold volume of ice-cold distilled underwent a process of acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated with a protein precipitation protocol in acetonitrile, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations from 625 to 320,000ng/mL (r(2)=0.998). Accuracy, intra-day and inter-day precision for ribavirin and phosphorylated-ribavirin quality controls were all below 9.0%. We tested this method by monitoring blood ribavirin concentrations in 13 HCV+ patients, receiving alpha interferon-plus ribavirin combination therapy.

Development and validation of a useful HPLC-UV method for quantification of total and phosphorylated-ribavirin in blood and erythrocytes of HCV+ patients

D'AVOLIO, ANTONIO
First
;
DE NICOLO', AMEDEO;SIMIELE, MARCO;BOGLIONE, Lucio;CUSATO, JESSICA;BAIETTO, LORENA;CALCAGNO, Andrea;SCIANDRA, Mauro;DI PERRI, Giovanni;BONORA, Stefano
Last
2012

Abstract

Ribavirin-induced hemolytic anemia is the main cause of discontinuation of the combination therapy with alpha-interferon-2b and ribavirin for the treatment of hepatitis C virus (HCV) infection. The determination of intracellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, which could be caused by the accumulation of these molecules within the erythrocytes. In this work, we simplified and validated a previously developed assay method, to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a five-fold volume of ice-cold distilled underwent a process of acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated with a protein precipitation protocol in acetonitrile, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations from 625 to 320,000ng/mL (r(2)=0.998). Accuracy, intra-day and inter-day precision for ribavirin and phosphorylated-ribavirin quality controls were all below 9.0%. We tested this method by monitoring blood ribavirin concentrations in 13 HCV+ patients, receiving alpha interferon-plus ribavirin combination therapy.
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http://dx.doi.org/10.1016/j.jpba.2012.03.030
A. D'Avolio;A. De Nicolò;M. Simiele;S. Turini;D. Agnesod;L. Boglione;J. Cusato;L. Baietto;G. Cariti;A. Calcagno;M. Sciandra;G. Di Perri;S. Bonora
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/110573
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